Bottom Line:
Quantitative analysis revealed that the induction of uropods results in a 5-10-fold increase in cell recruitment.Additional studies showed that the cell recruitment mediated by uropods was abrogated with antibodies to ICAM-1, -3, and LFA-1, whereas mAb to CD43, CD44, CD45, and L-selectin did not have a significant effect, thus indicating that the interaction of LFA-1 with ICAM-1 and -3 appears to be responsible for this process.An enhancement of T cell migration was observed under conditions of uropod formation, and this increase was prevented by incubation with either blocking anti-ICAM-3 mAbs or drugs that impair uropod formation.

ABSTRACTThe recruitment of leukocytes from the bloodstream is a key step in the inflammatory reaction, and chemokines are among the main regulators of this process. During lymphocyte-endothelial interaction, chemokines induce the polarization of T lymphocytes, with the formation of a cytoplasmic projection (uropod) and redistribution of several adhesion molecules (ICAM-1,-3, CD43, CD44) to this structure. Although it has been reported that these cytokines regulate the adhesive state of integrins in leukocytes, their precise mechanisms of chemoattraction remain to be elucidated. We have herein studied the functional role of the lymphocyte uropod. Confocal microscopy studies clearly showed that cell uropods project away from the cell bodies of adhered lymphocytes and that polarized T cells contact other T cells through the uropod structure. Time-lapse videomicroscopy studies revealed that uropod-bearing T cells were able, through this cellular projection, to contact, capture, and transport additional bystander T cells. Quantitative analysis revealed that the induction of uropods results in a 5-10-fold increase in cell recruitment. Uropod-mediated cell recruitment seems to have physiological relevance, since it was promoted by both CD45R0+ peripheral blood memory T cells as well as by in vivo activated lymphocytes. Additional studies showed that the cell recruitment mediated by uropods was abrogated with antibodies to ICAM-1, -3, and LFA-1, whereas mAb to CD43, CD44, CD45, and L-selectin did not have a significant effect, thus indicating that the interaction of LFA-1 with ICAM-1 and -3 appears to be responsible for this process. To determine whether the increment in cell recruitment mediated by uropod may affect the transendothelial migration of T cells, we carried out chemotaxis assays through confluent monolayers of endothelial cells specialized in lymphocyte extravasation. An enhancement of T cell migration was observed under conditions of uropod formation, and this increase was prevented by incubation with either blocking anti-ICAM-3 mAbs or drugs that impair uropod formation. These data indicate that the cell interactions mediated by cell uropods represent a cooperative mechanism in lymphocyte recruitment, which may act as an amplification system in the inflammatory response.

Figure 2: Lymphocyte uropods promote cell contacts between T lymphoblasts. Confocal microscopy analysis of lymphocyte–lymphocyte interactions mediated by cell uropods. T lymphoblasts labeled with the green fluorescent cytoplasmic probe CFDA-SE were allowed to bind to ICAM-1–Fc–coated coverslips (10 μg/ml) for 30 min at 37°C in the presence of the uropod inducing anti–ICAM-3 HP2/19 mAb. Cells were then fixed and stained for ICAM-3 (red fluorescence). Slides were analyzed as described in Materials and Methods. Optical sections were adjusted to the plane of adhesion (A and B) or 5 μm above (C and D). E corresponds to the three-dimensional reconstruction of cells, simultaneously showing the cytoplasmic (green) and ICAM-3 (red) staining.

Mentions:
The projection of uropod towards outer milieu and its high content of several adhesion molecules further suggested its involvement in the recruitment of free lymphocytes. To address this point we carried out a confocal microscopy analysis of the contacts that occur between adhered, uropod-bearing lymphocytes and other T cells. Interestingly, we found that the anchorage between those cells was located 5 μm above of the plate level and that it was mediated by the ICAM-3–bearing uropod of one cell (Fig. 2, A–D). Three-dimensional reconstruction of all different optical sections of the specimen and rotation of the image clearly confirmed that the intercellular contact was mediated by the uropod (Fig. 2 E). Similar cell–cell contacts were observed when the uropod was induced by the relevant physiological stimuli, the chemokines (Fig. 1 b).

Figure 2: Lymphocyte uropods promote cell contacts between T lymphoblasts. Confocal microscopy analysis of lymphocyte–lymphocyte interactions mediated by cell uropods. T lymphoblasts labeled with the green fluorescent cytoplasmic probe CFDA-SE were allowed to bind to ICAM-1–Fc–coated coverslips (10 μg/ml) for 30 min at 37°C in the presence of the uropod inducing anti–ICAM-3 HP2/19 mAb. Cells were then fixed and stained for ICAM-3 (red fluorescence). Slides were analyzed as described in Materials and Methods. Optical sections were adjusted to the plane of adhesion (A and B) or 5 μm above (C and D). E corresponds to the three-dimensional reconstruction of cells, simultaneously showing the cytoplasmic (green) and ICAM-3 (red) staining.

Mentions:
The projection of uropod towards outer milieu and its high content of several adhesion molecules further suggested its involvement in the recruitment of free lymphocytes. To address this point we carried out a confocal microscopy analysis of the contacts that occur between adhered, uropod-bearing lymphocytes and other T cells. Interestingly, we found that the anchorage between those cells was located 5 μm above of the plate level and that it was mediated by the ICAM-3–bearing uropod of one cell (Fig. 2, A–D). Three-dimensional reconstruction of all different optical sections of the specimen and rotation of the image clearly confirmed that the intercellular contact was mediated by the uropod (Fig. 2 E). Similar cell–cell contacts were observed when the uropod was induced by the relevant physiological stimuli, the chemokines (Fig. 1 b).

Bottom Line:
Quantitative analysis revealed that the induction of uropods results in a 5-10-fold increase in cell recruitment.Additional studies showed that the cell recruitment mediated by uropods was abrogated with antibodies to ICAM-1, -3, and LFA-1, whereas mAb to CD43, CD44, CD45, and L-selectin did not have a significant effect, thus indicating that the interaction of LFA-1 with ICAM-1 and -3 appears to be responsible for this process.An enhancement of T cell migration was observed under conditions of uropod formation, and this increase was prevented by incubation with either blocking anti-ICAM-3 mAbs or drugs that impair uropod formation.

ABSTRACTThe recruitment of leukocytes from the bloodstream is a key step in the inflammatory reaction, and chemokines are among the main regulators of this process. During lymphocyte-endothelial interaction, chemokines induce the polarization of T lymphocytes, with the formation of a cytoplasmic projection (uropod) and redistribution of several adhesion molecules (ICAM-1,-3, CD43, CD44) to this structure. Although it has been reported that these cytokines regulate the adhesive state of integrins in leukocytes, their precise mechanisms of chemoattraction remain to be elucidated. We have herein studied the functional role of the lymphocyte uropod. Confocal microscopy studies clearly showed that cell uropods project away from the cell bodies of adhered lymphocytes and that polarized T cells contact other T cells through the uropod structure. Time-lapse videomicroscopy studies revealed that uropod-bearing T cells were able, through this cellular projection, to contact, capture, and transport additional bystander T cells. Quantitative analysis revealed that the induction of uropods results in a 5-10-fold increase in cell recruitment. Uropod-mediated cell recruitment seems to have physiological relevance, since it was promoted by both CD45R0+ peripheral blood memory T cells as well as by in vivo activated lymphocytes. Additional studies showed that the cell recruitment mediated by uropods was abrogated with antibodies to ICAM-1, -3, and LFA-1, whereas mAb to CD43, CD44, CD45, and L-selectin did not have a significant effect, thus indicating that the interaction of LFA-1 with ICAM-1 and -3 appears to be responsible for this process. To determine whether the increment in cell recruitment mediated by uropod may affect the transendothelial migration of T cells, we carried out chemotaxis assays through confluent monolayers of endothelial cells specialized in lymphocyte extravasation. An enhancement of T cell migration was observed under conditions of uropod formation, and this increase was prevented by incubation with either blocking anti-ICAM-3 mAbs or drugs that impair uropod formation. These data indicate that the cell interactions mediated by cell uropods represent a cooperative mechanism in lymphocyte recruitment, which may act as an amplification system in the inflammatory response.